Assessment of Antibody Stability in a Novel Protein-Free Serum Model

Abstract

Therapeutic proteins can degrade upon administration as they are subjected to a variety of stresses in human body compartments. In vivo degradation may cause undesirable pharmacokinetic/pharmacodynamic profiles. Pre-clinical in vitro models have gained scientific interest as they enable one to evaluate the in vivo stability of monoclonal antibodies (mAbs) and ultimately can improve patient safety. We used a novel approach by stripping serum of endogenous proteins, which interfere with analytical test methods. This enabled the direct analysis of the target protein without laborious sample work-up procedures. The developed model retained the osmolality, conductivity, temperature, and pH of serum. We compared the impact of human, bovine, and artificial serum to accelerated stability conditions in histidine buffer. Target mAbs were assessed in regard to visible and sub-visible particles, as well as protein aggregation and fragmentation. Both mAbs degraded to a higher extent under physiological conditions compared to accelerated stability conditions. No relevant stability differences between the tested mAbs were observed. Our results reinforced the importance of monitoring protein stability in biological fluids or fluids emulating these conditions closely. Models enabling analysis in fluids directly allow high throughput testing in early pre-clinical stages and help in selecting molecules with increased in vivo stability.

Combined Presence of Ferrous Ions and Hydrogen Peroxide in Normal Saline and In Vitro Models Induces Enhanced Aggregation of Therapeutic IgG due to Hydroxyl Radicals

“…Normal saline is isotonic and consists of 0.9% NaCl . However, once the sample containing mAb is administered in the body, it is exposed to various salts, pH of 7.4, and different small and large molecules. ,− Hence, to determine the aggregation of mAb samples with FeSO 4 ·7H 2 O and H 2 O 2 after administration in the body, stability was investigated in extracellular saline and more complex in vitro models.…”

“…Once the formulation of therapeutic IgG is administered in the body, the excipients will get diluted in the body fluids and the mAb would be exposed to different chemicals. , However, physiological fluid such as serum has a pH of 7.4 maintained by the carbonate–bicarbonate buffer , and has various salts, proteins, and small and large molecules. , Hence, the aggregation of mAb was explored in extracellular saline having a physiological pH and different ions other than Na + , and it was found that the sample in the combined presence of H 2 O 2 and Fe 2+ resulted in rapid aggregation of mAb.…”

Protein Aggregates in Inhaled Biologics: Challenges and Considerations

“…It is important to note that the guidance for sub-visible particles is not designed to de-risk protein aggregation in vivo or immunogenicity concerns, but rather to avoid the risk of capillary occlusion 27 . Pre-clinical in vitro models are increasingly employed to evaluate the protein stability post injection 28 , 29 , 30 . For instance, a novel protein-free serum in vitro setup revealed faster degradation profiles for two monoclonal antibodies (mabs) compared to accelerated stability studies 30 .…”

“…Pre-clinical in vitro models are increasingly employed to evaluate the protein stability post injection 28 , 29 , 30 . For instance, a novel protein-free serum in vitro setup revealed faster degradation profiles for two monoclonal antibodies (mabs) compared to accelerated stability studies 30 . However, the guidance for particulate levels serves as a unified regulation across parenteral formulations to limit particles, including protein aggregates, in the final product.…”

Pre-Clinical In-Vitro Studies on Parameters Governing Immune Complex Formation

“…Alternatively, the assessment of IC formation using degraded biologics material from incubation in biomatrices like serum could be an attractive option. Due to their extended half-life, many biologics are known to experience modifications from being exposed to elevated temperatures and to serum components [46].…”